The vitamin D5 series of compounds are different from the D3 series. In vitamin D5, there is an ethyl group at the C-24 position. Earlier, we showed that an analog of vitamin D5, 1alpha-Hydroxyvitamin D5 (1alpha(OH)D5), is relatively nontoxic and non-calcemic compared to other vitamin D analogs (1). During the past 2 years of the funding period, we have shown that 1alpha(OH)D5 was effective in inhibiting both the tumor incidence and multiplicity of MNU- and DMBA-induced mammary carcinogenesis in rats. We also showed that the effect of 1alpha(OH)D5 was more prominent during the promotion phase of carcinogenesis. Here we will evaluate efficacy of 1alpha(OH)D5 in clinically relevant experimental protocol either singly or in combination with tamoxifen. More recently, the gene array profile of normal and transformed human mammary epithelial cells suggested that, among the differentially expressed genes, prohibitin, a cell cycle regulatory gene was upregulated in transformed cells. Moreover, prohibitin was down-regulated when transformed cells were treated with 1alpha(OH)D5. This provides a clue for understanding D5-mediated cell cycle regulation in transformed cells. We hypothesize that, in addition to vitamin D receptor, prohibitin may be an essential target for the action of 1alpha(OH)D5 in chemoprevention. Very little has been reported regarding the metabolism and pharmacokinetics of the D2, D4, and D5 series of vitamin D analogs. Recently, Jones and colleagues reported that C24 methyl or ethyl might be a metabolic determinant for vitamin D4 and D5 analogs (2). Dr. Glenville Jones will collaborate with us on this project to evaluate metabolism of 1alpha(OH)D5 in normal and neoplastic breast cells and tissues. Understanding of both the mechanism of action and metabolism of 1alpha(OH)D5 is crucial in developing this analog for clinical application. The following specific aims are proposed to determine: 1. Whether 1alpha (OH)D5 prevents development of tumors subsequent to surgical excision of the first palpable tumor? Whether the combination of 1alpha(OH)D5 and tamoxifen provide enhanced protection compared to either agent alone? 2. Whether prohibitin plays a role in cell transformation and chemopreventive efficacy in these cells? 3. How is 1alpha(OH)D5 metabolized in normal and neoplastic mammary epithelial cells and tissues?